Western Blot Troubleshooting | Antibody Company, Buy Antibodies Online (2024)

Pro tips on resolving common Western Blot issues such as weak signal, wrong band size, smiley gel, and high background.

How to Troubleshoot Western Blot

The following guide serves as a checklist for the possible causes and solutions to some of the most commonly encountered problems with Western blot assays.

If you do not see the issues you are having featured in this page, please contact us at [emailprotected] and we will help you resolve your specific trouble.

Troubleshooting Guides

Download troubleshooting handbooks for IHC, Western blot, and ELISA for FREE.

Troubleshooting Guides

Are you having Troubleshooting issues with Western Blotting?

Let Bosterbio handle your Western blot CRO services and be your long term partner in broadening the bandwidth of your lab/team in a flexible manner.

Learn more about Western Blot CRO services!

High Background

High background on a western blot occurs when the background signal of the membrane reduces the signal-to-noise ratio to unreadable levels. Typically, polyacrylamide gel electrophoresis separates proteins by molecular weight, with large proteins migrating slower than small proteins. This process can be affected by several factors: protein degradation can cause proteins to appear shorter than the expected length, glycosylation can cause proteins to appear larger than predicted, and nonspecific antibody binding can cause multiple bands to appear on a blot where only one is expected.

Use these tips to identify and resolve the source of your unexpected band sizes.

S.No.	Possible Cause	Solution
1	Antibody concentration is too high	Optimize and decrease antibody concentration Use a dot-blot test to optimize antibody concentration
2	Aggregate secondary antibody formation	Filter the secondary antibody through 0.2μm filter Use a new secondary antibody
3	Too high antibody incubation temperature	Incubate the antibody at 4°C
4	Non-specific secondary antibody binding or cross-reactivity with blocking agent	Run secondary antibody control (without the primary) Decrease secondary antibody concentration
5	Cross-reactivity of primary or secondary antibody with blocking agent	Add Tween-20 to the incubation and washing buffer
6	Incompatible blocking agent	Compare different blocking buffers
7	Incomplete blocking	Optimize choice of blocking buffer Increase protein concentration in blocking agent Optimize blocking time and/or temperature; Block for 2 hours at normal temperature or overnight at 4°C Add 0.05% Tween 20 detergent into blocking agent Add 0.05% Tween 20 detergent into antibody diluents solution
8	Insufficient blocking	Extend blocking time, increase blocking solution concentration, or use a compatible blocking agent (e.g. skim milk, BSA, serum, etc.)
9	Cross-reactivity of antibody with other proteins	Use different blocking agent (Do not use skim milk with biotin system Reduce secondary antibody concentration Test cross-reactivity between secondary antibody and membrane
10	Insufficient washing	Increase number of washes and buffer volume Add 0.05% Tween 20 detergent into washing buffer
11	Exposure time is too long	Check exposure parameters and reduce exposure time
12	Membrane problem	Use clean tweezers; Operate with gloves Use new membranes Ensure the liquid is enough to keep the membrane moist Use decolorization table in incubation Avoid membranes overlapping Handle carefully and avoid damaging membrane
13	Insufficient membrane wash	Increase the number of wash
14	Incompatible membrane	Nitrocellulose membrane’s background is lower than that of PVDF membrane
15	Dry membrane	Make sure membrane is covered with a sufficient amount of liquid and prevent it from drying
16	Contaminated buffer	Use new buffer or filter buffer before use Never re-use blocking solutions Use pure protein as a blocking agent
17	Contaminated equipment	Ensure all equipment and tools are clean and no gel is left on membrane Use disposable incubation trays Thoroughly wash reusable incubation trays between incubations
18	Insufficient antibody binding activity	Use fresh aliqot of antibody stored at -20°C Avoid thawing and re-freezing antibodies Store antibodies at -80°C for long-term stability
19	Excessive substrate incubation	Reduce length of substrate incubation
20	Blocking proteins reacting with detection system	Milk contains biotin; do not use when detecting with avidin-biotin system Try one of Boster's other blocking reagents

Weak/No Signal

A weak western blot signal is characterized by faint or indistinct bands. While the bands may be barely visible at their predicted sizes, weak signal can often require repeating the experiment. Common sources of this error occur during blocking, washing, protein transfer, or detection.

Use the tips below to identify the source of the error and get better results.

S.No.	Possible Cause	Solution
1	Improper protein transfer to membrane	Stain gel after transfer is complete to determine transfer is efficient Use Ponceau S to stain membrane to determine transfer is efficient Ensure sufficient contact between gel and membrane during transfer Make sure transfer sandwich is assembled correctly Wet membrane according the instruction Avoid overheating during electro-transfer Use positive control or molecular weight markers Optimize transfer time and current Use Boster’s Membrane-Transferring Buffer (AR1149) Avoid sample (antigenic determinant) destroy when handling
2	Insufficient protein and membrane binding	Adding 20% methanol to transfer buffer Use small-bore membrane
3	Insufficient antibody	Increase antibody concentration
4	Insufficient antigen	Load more protein
5	Antigen masking by blocking buffer	Compare different blocking buffers Optimize protein concentration of blocking agent Reduce blocking time Nonfat dry milk can sometimes mask antigens. Try using a different blocking reagent.
6	Presence of sodium azide in buffers	Eliminate sodium azide from buffers
7	Too short exposure time	Lengthen film exposure time
8	Too short substrate incubation time	Lengthen substrate incubation time to five minutes
9	Digestion of protein on membrane	Optimize amount of blocking agent
10	Degradation of protein during storage	Re-prepare protein sample
11	Incompatible primary and secondary antibodies	Make sure primary antibody, secondary antibody, substrate, enzyme system and samples are compatible Use loading control to test effectiveness of second detecting system
12	Low concentration of primary antibody and/or secondary antibody	Increase antibody concentration Increase incubation time Use a dot blot assay to optimize protein concentration
13	Cross-reactivity between blocking agent and antibodies (primary or secondary)	Use mild detergent such as Tween20 Change blocking agent (commonly used are milk, BSA, serum or gelatin)
14	Inability of primary antibody to recognize the protein in tested sample	Check instruction Use positive control
15	Low or none content of target protein (ineffective antigen)	Use positive control Increase loading amount to 20-30 µg protein per well Use protease inhibitor or fractional extract target protein
16	Insufficient transfer and excessive wash	Check the transfer with Ponceau S Soak PVDF-membrane in methanol Avoid excessive wash
17	Over-blocking	Use 0.05% skim milk or no milk diluents buffer Change blocking agent Reduce blocking time
18	Loss of primary antibody effectiveness	Prepare fresh antibody and store properly when not in use Avoid repeated freezing and thawing
19	Inhibition of secondary antibody by sodium azide	Avoid using sodium azide together with HRP- conjugated antibodies
20	Loss of effectiveness in enzyme conjugate and substrate	Mix enzyme conjugate and substrate (no color development when enzyme is inactive) Use activated enzyme conjugate and fresh substrate
21	Improper wet transfer for membrane	Soak PVDF membrane in 100% methanol
22	Insufficient molecular weight of target protein (< 10 kDa)	Use small-bore membrane Reduce transfer time
23	Equality or nearness in values between target protein’s isoelectric point and transfer buffer’s pH value	Try other buffers such as CAPS buffer (pH 10.5) Try low pH value buffers such as acetic acid buffer
24	Too high methanol concentration	Decrease methanol concentration or use isopropyl alcohol
25	Insufficient sample concentration	Increase the amount of starting material Concentrate your sample using immunoprecipitation or similar procedure
26	Transfer too vigorous	Reduce transfer time or voltage to prevent small proteins transferring completely through membrane Use a secondary membrane to capture proteins transferred through the primary membrane Use a membrane with smaller pore size
26	Inadequate transfer	Increase transfer time or voltage
27	Sandwich assembly oriented incorrectly	Make sure the sandwich assembly is oriented correctly relative to the electric field Check the polarity of the electric field
28	Incorrect transfer buffer pH	Adjust transfer buffer PH to be 2 points lower than the pI of protein sample to optimize charge:mass ratio
29	Insufficient antibody binding affinity	Reduce washing stringency Increase antibody concentration Use Boster high affinity primary antibodies
30	Insufficient sample loading	Use more starting material Concentrate sample prior to loading

Blotchy, Flecked, Or Dirty Background

The background of a western blot does not always appear clean and flawless. Blotches, streaks, and spots are all common artifacts that can make it hard to interpret and publish your results. These artifacts are most commonly the result of uneven coating of buffer or antibody, the membrane drying out, or aggregates forming in the antibody or blocking buffer.

Follow the tips below to identify and solve the cause of your imperfect western blot background.

S.No.	Possible Cause	Solution
1	Blotched background: Uneven antibody distribution	Agitate during incubation to coat the membrane evenly in incubation buffer
2	Blotched background: Membrane dried out unevenly	Make sure membrane is thoroughly wetted before starting the protocol Ensure the membrane does not dry out during any step
3	Blotched background: Uneven wash/incubation buffer coverage	Increase volume of wash and incubation buffers Do not stack membranes during incubation
4	Flecked background: Secondary antibody aggregation	Increase secondary antibody dilution to prevent aggregation Spin down or filter out antibody aggregates
5	Flecked background: Clumps of blocking buffer binding secondary antibody	Use fresh blocking buffer Increase Tween 20 concentration of blocking buffer Filter blocking buffer before use Use a different blocking reagent, such as albumin, BSA, or casein Wash membrane with wash buffer before antibody incubation
6	Flecked background: Buffer contamination	Mix new buffers Filter buffers before use
7	White spots with no protein transfer: Air bubbles trapped between gel and membrane during transfer	Carefully squeeze out bubbles from between membrane and gel using a sterile glass rod Use enough buffer to saturate the membrane

Wrong Band Sizes

Typically, polyacrylamide gel electrophoresis separates proteins by molecular weight, with large proteins migrating slower than small proteins. This process is can be affected by several factors: protein degradation can cause proteins to appear shorter than the expected length, glycosylation can cause proteins to appear larger than predicted, and nonspecific antibody binding can cause multiple bands to appear on a blot where only one is expected.

Use these tips to identify and resolve the source of your unexpected band sizes.

S.No.	Possible Cause	Solution
1	Bands have higher MW than expected: Proteins are glycosylated or bear other post-translational modifications	Review literature and identify modified forms of your target protein Strip post-translational modifications with enzymatic treatment
2	Bands have much higher MW than expected: Protein aggregation	Decrease protein concentration Prepare new sample with fresh loading buffer
3	Bands have much higher MW than expected: Incomplete denaturation or residual disulfide bonding	Denature the protein with urea Use stronger reducing agents Use fresh 2-mercaptoethanol or DDT to strip disulfide bonds
4	Bands have lower MW than expected: Protein sample has been digested or degraded	Use fresh sample from frozen stock Use a lysis buffer with proteinase inhibitors
5	Bands have lower MW than expected: Primary antibody is detecting splice variants	Review literature to identify splice variants of your protein Try a different primary antibody
6	Bands have lower MW than expected: Primary antibody binding a similar epitope on a different protein	Run a negative control to detect other proteins that react with your antibody
7	Multiple bands: Primary or secondary antibody contaminated with nonspecific IgG	Use Boster primary antibodies guaranteed free of nonspecific IgG
8	Multiple bands: Nonspecific binding of primary antibody	Increase antibody dilution Affinity purify primary antibody to select for only desired binding activity Use Boster primary antibodies guaranteed to only bind their indicated targets
9	Multiple bands: Nonspecific binding of secondary antibody	Reduce antibody concentration Run a negative control with just the secondary antibody to detect nonspecific binding
10	Multiple bands: Insufficient blocking	Use higher concentration blocking buffer Block for longer Add Tween 20 to blocking buffer
11	Multiple bands: Ionic interactions	Increase stringency of washing step Increase salt concentration of incubation buffers Include stronger detergents in the washes

Distorted Bands

Distorted bands can make it very hard to interpret your results. Common distortions include smile-shaped bands with the edges trailing upward, diffuse bands that are broad or blurry, and streaked bands that trail off in several directions.

Make sure your next blot has even, crisp bands by following the tips below.

S.No.	Possible Cause	Solution
1	Curved, "smiling" bands: Electrophoresis voltage too high	Reduce voltage; run the gel slower for more consistent results
2	Curved, "smiling" bands: Overheated gel	Reduce voltage or run the gel on ice or in a cold room (-4°C)
3	Streaked or diffuse bands: Incomplete contact between gel and membrane during transfer	Use thicker filter paper in sandwich Squeeze air bubbles and excess buffer from between membrane and gel
4	Streaked or diffuse bands: Slippage of membrane during transfer	Avoid agitating or moving the gel or membrane during transfer
5	Blurry bands: Electrophoresis voltage too high	Run the gel for longer at a lower voltage
6	Blurry bands: Improper loading buffer composition	Mix new loading buffer, or use Boster formulated loading buffer
7	Blurry bands: Air bubbles trapped between gel and membrane during transfer	Carefully remove bubbles by squeezing them out with a sterile glass rod
8	Ghost bands: Overexposure during visualization	Decrease the exposure time
9	Ghost bands: Loading sample too concentrated	Reduce the amount of sample loaded
10	Ghost bands: Antibody concentration too high	Dilute your antibody solutions using Boster's antibody diluent medium
11	Ghost bands: Blot was moved during transfer	Avoid agitating or moving the gel or membrane during transfer
12	Uneven, crooked bands: Poor gel polymerization	Check gel concentration and ensure complete dissolution of SDS before casting gels
13	Uneven, crooked bands: Varying salt concentration between wells	Make sure the salt concentration in different samples is similar

Unlock the full potential of your research and extract invaluable insights from your samples with our exceptional service. Don't hesitate—contact us now to discuss your project requirements and see how our expert Western blotting service can elevate your research.

Western Blotting Principle

Learn the concept behind Western blotting. It is a technique that is used to detect specific proteins in the given sample. It usually involves two major processes, namely, SDS-polyacrylamide gel electrophoresis and protein blotting and testing.

Learn about Western Blot Principle

Western Blot Sample Preparation

Check out this Western Blot sample preparation guides to learn how to get the best results from your sample type. Learn more about sample preparation in this guide.

Learn Western Blot Sample Preparation

Western Blot Protocol

Learn a stepwise Western blotting protocol from reagent preparation to detection with application of BosterBio reagents. Check out our ELISA protocols to learn how to get the best results.

Check our Western Blot Protocol

Western Blotting Optimization

Get to learn the concept behind our best practises on Western Blot optimization. Learn how to optimize every aspect of your experiment to yield the best results.

Browse Western Blot Optimization Tips

Western Blot Troubleshooting | Antibody Company, Buy Antibodies Online (2024)

FAQs

What are the problems with western blot? ›

Weak or no signal

Possible cause	Solutions
Blocking buffer blocks antigen	Evaluate another blocking buffer.
Quantity of sample loaded on the gel	Too much lysate can overcrowd your specific target and reduce the antibody sensitivity.
Quantity of sample loaded on the gel	Too little lysate leads to insufficient availability of the target of interest.

17 more rows

Tell Me More ›

How do you find the best antibody for western blot? ›

Choose antibodies designated specifically for western blotting or that list western blotting as an application. In addition, it is important to confirm that the antibody is specific towards the native or denatured protein, to determine if SDS-PAGE or native PAGE should be performed.

Why would an antibody not work? ›

Antibody-related issues can arise due to various factors, such as the antibody's functionality, experimental conditions, non-specific bindings, concentration, storage, compatibility with the detection method, and previous experiences of others.

Tell Me More ›

Can I reuse antibody for western blot? ›

In general, you'll see only a slight difference in band intensity and blot quality obtained with freshly diluted and reused primary antibodies. Pay attention to how many times the antibody solution has been used and dispose of it when the signal begins to fade.

Explore More ›

Why is western blot no longer used? ›

The HIV-1 Western blot (WB), the historic gold standard for laboratory diagnosis of HIV-1 infection, is no longer part of the recommended algorithm. The two main reasons for this are the inability of the WB to detect acute infection and the potential to misclassify HIV-2 infection as an HIV-1 infection.

Know More ›

What are the sources of error in the western blot test? ›

The secondary antibody can sometimes react with a non-intended protein, and this can cause the labeling of an incorrect protein. [39] Insufficient transfer time can result in the larger proteins not transferring correctly. This can cause erroneous bands or no bands at all.

Find Out More ›

How to determine which antibody to use? ›

To successfully choose a secondary antibody, one that is best for your application and research, consider the following factors:

Host and target species.
Targeted reactivity.
Purification.
Cross-adsorption.
Multiplexing.
Antibody class and subclass.
Whole antibodies vs. fragments.
Conjugates.

More items...

Learn More Now ›

How to choose antibodies? ›

7 Tips for Selecting the Perfect Antibody

Choosing the right target of interest. ...
Primary or secondary antibodies? ...
Species compatibility. ...
Formulation and degree of purification. ...
Antibody and application validation. ...
Multiplexing. ...
Supplier-provided resources.

Jan 9, 2024

What happens if you don't have enough antibodies? ›

People with a deficiency should also expect more inflammation, like more swelling and more severe fevers, and a longer time to recover from illness. “If a person has an antibody deficiency, that means that the person might have a higher risk of an increased frequency of infections,” he said.

Read The Full Story ›

What is the most common antibody deficiency? ›

The most common antibody deficiency disorders include:

X-linked agammaglobulinemia (Bruton disease)
Transient hypogammaglobulinemia of newborn.
Selective Ig immunodeficiencies, for example, IgA selective deficiency.
Super IgM syndrome.
Common variable immunodeficiency disorder.

May 23, 2023

Show Me More ›

What are bad antibodies? ›

The 'bad' antibodies are known to react against the body's own tissues and can cause autoimmune disease. For this reason, it was once thought that they were discarded by the immune system or that they were made inactive in the long term.

Get More Info ›

How long do antibodies last in fridge? ›

In most cases, storage at 4°C upon receipt of the antibody is acceptable for one to two weeks. Make sure not to use a frost-free freezer: it's unlikely your lab would, but the cycling between freezing and thawing should be avoided.

Discover More Details ›

Do antibodies go bad? ›

Antibodies are relatively stable proteins and are resistant to a broad range of mild denaturing conditions. Most antibodies are stable for years when stored properly as per manufacturer's recommendations. In most cases antibodies can be stored at -20 °C without any loss in their binding capacity.

Can you clone an antibody? ›

Alternative approaches have since been developed to isolate memory B cells from peripheral blood and then isolate and clone the antibody variable regions using single-cell expression cloning [30].

Find Out More ›

What is the disadvantage of Western blot test? ›

The main disadvantage of Western blotting is that this technique requires a specific antibody to a target protein; thus many protein targets cannot be investigated because of the lack of specific antibodies.

What are the pros and cons of Western blotting? ›

Advantages of Western blotting include the ability to identify specific target proteins, while disadvantages include difficulties in reproducibility and time-consuming procedures.

View Details ›

What are the false negatives in Western blotting? ›

False negatives may be caused by physical loss of a conformational epitope or by post-translational modifications that mask the epitope. Building a successful assay is then down to the scientist, and those pesky laws of physics!

What are the limitations of blotting techniques? ›

2.2 Limitations of Blotting. Blotting is more time- and labor-intensive than newer techniques and probably not as sensitive. The technique and reagents selected can determine the specificity and sensitivity of blotting. Verification of the quantity of molecules present is not as precise as with newer techniques.

Western Blot Troubleshooting | Antibody Company, Buy Antibodies Online (2024)

How to Troubleshoot Western Blot

Troubleshooting Guides

Are you having Troubleshooting issues with Western Blotting?

High Background

Weak/No Signal

Blotchy, Flecked, Or Dirty Background

Wrong Band Sizes

Distorted Bands

Related Pages

Western Blotting Principle

Western Blot Sample Preparation

Western Blot Protocol

Western Blotting Optimization

FAQs

What are the problems with western blot? ›

What are bad antibodies? ›